BACKGROUND This article reports a method to obtain vascular smooth muscle cells (SMCs) from the spiral modiolar artery (SMA) of guinea pigs and provides materials for related experimental studies. MATERIAL AND METHODS SMA was separated from the cochlea of guinea pigs, digested with trypsin (1.25 g/L) and allowed to adhere in a 35-mm culture dish. The morphology of the sample was investigated, and the sample was identified by immunofluorescence analysis, flow cytometry, Western blot, and RT-PCR. Cell viability was calculated using trypan blue and flow cytometry. Whole-cell patch clamp was used to record the membrane input resistance (Rinput), reciprocal membrane input conductance (Ginput), membrane input capacitance (Cinput), and resting membrane potential (RP) of the SMCs. RESULTS Microscopy results showed that the cells had typical peak-valley growth pattern. The cell growth curve was similar to an S curve, and flow cytometry results showed that the cell apoptosis rate was less than 10%. Moreover, flow cytometry, immunofluorescent staining, Western blot and RT-PCR detected the specific and intensely positive expression of cell type-specific markers α-SM-actin, SM22α, calponin and desmin. Furthermore, following properties of the P3 and P6 cells were obtained: Rinput, 2611±356 and 2477±338 MΩ; Ginput, 0.454±0.071 and 0.273±0.037 ns; Cinput, 17.029±0.917 and 18.042±1.051 pF, and RP -20.602±1.503 and -22.192±1.905 mV. CONCLUSIONS Various highly purified SMCs were obtained from the SMA of guinea pigs. We provide an ideal experimental material for the study of the pathogenesis of diseases related to the circulation disturbances in the inner ear in vitro. The results can be used to evaluate the effects of drugs on vascular smooth muscle.